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Review
Activating Drugs with Sound: Mechanisms Behind Sonodynamic
Therapy and the Role of Nanomedicine
30th Anniversary Review
Victor Choi,# Maneesha A. Rajora,# and Gang Zheng*
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ABSTRACT: Sonodynamic therapy (SDT) is a promising therapeutic platform for
minimally invasive cancer treatment in which acoustically susceptible drug agents,
sonosensitizers, are activated by deep-tissue-penetrating low frequency ultrasound.
Despite growing research in recent years, the field has yet to clearly elucidate broadly
applicable mechanisms by which acoustic cavitation triggers sonosensitizer therapeutic
activity, creating difficulties in achieving substantial and translatable therapeutic efficacy.
In this review, we will critically analyze the proposed mechanisms underlying SDT and
overview how nanomedicines can complement and extend these mechanisms to deliver
more efficacious SDT. In doing so, we aim to highlight potential avenues toward viable
implementation of SDT as a cancer therapy.
1. INTRODUCTION TO SONODYNAMIC THERAPY: A
LOW FREQUENCY BIOMEDICAL APPLICATION OF
ULTRASOUND
The biomedical applications of ultrasound have evolved from
its ubiquitous clinical use as an imaging tool to include an
expanding array of therapeutic capabilities for the treatment of
cancer. This arena of therapeutic ultrasound is rooted in the
delivery of low (∼1 MHz)1 frequency pressure pulses to tissues
at depths that can exceed 10 cm.2 These sound waves can be
delivered in a focused manner, guided by imaging modalities
such as magnetic resonance imaging (MRI), to target tissue.
The energy deposited can translate into the generation of heat
for tumor ablation, giving rise to high intensity focused
ultrasound (HIFU)3 thermal ablation, or be pulsed at high
intensities for nonthermal tissue fractionation via histotripsy,4
both of which serve as alternatives or adjuvants for conventional cancer treatment. Alternatively, at lower intensities,
therapeutic ultrasound can generate largely nonthermal
bioeffects when coadministered with microbubbles. These
gas bubbles, stabilized by a lipid, polymer, or protein shell, are
typically vascular agents that respond to the applied low
frequency ultrasound by expanding and contracting. This
phenomenon of microbubble oscillation,3 termed acoustic
cavitation, has been widely exploited to enhance the
extravasation and delivery of chemotherapeutic drugs to
deep-seated target lesions, including beyond the blood-brain
barrier to glioblastomas preclinically and clinically, as well as to
pancreatic tumors, the liver and kidneys preclinically.5−10
© XXXX American Chemical Society
Notably, the combined use of microbubble cavitation and a
focused nonionizing energy source has made focused low
intensity therapeutic ultrasound an attractive modality to
enhance chemotherapy delivery to deep tissues in a safe,
minimally invasive, controlled, and targeted manner.11
A less explored, but promising therapeutic application of low
frequency, low intensity therapeutic ultrasound is the acoustic
activation of drug agents, termed sonodynamic therapy.
Sonodynamic therapy (SDT) is loosely defined as the acoustic
activation of drug agents, termed sonosensitizers, by
coadministered low frequency ultrasound. This sonosensitizer
acoustic activation yields localized cytotoxicity, often attributed
to the generation of reactive oxygen species (ROS). First
explored in the 1990s,12 SDT originated as a proxy for
photodynamic therapy (PDT), a minimally invasive treatment
paradigm in which light is used to excite photoactive agents
(photosensitizers) to a triplet state that subsequently interacts
with oxygen and biomolecules to generate ROS.13 Both SDT
and PDT thus bear similar abilities to address unresectable
tumors, the invasiveness of surgery, and the off-target effects of
ionizing radiation and chemotherapy by delivering dualReceived: January 15, 2020
Revised: March 4, 2020
Published: March 4, 2020
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Figure 1. Acoustic cavitation of microbubbles. A driving pressure wave can breach fluid/solid boundaries or imperfections within a fluid, creating a
gas pocket during rarefaction that serves as cavitation nuclei. Alternatively, these nuclei can be present as small undissolved gas bubbles in biological
media or be administered as preformed exogenous microbubbles/ultrasound contrast agents. During compression, inrushing fluid compresses these
endogenous or exogenous microbubbles, causing gas to be dispelled to form a smaller-sized bubble. During rarefaction, gas enters the bubble as
fluid is pulled away causing the bubble to grow in size. Together, these cycles of compression and rarefaction create microbubble oscillations, which
below a bubble-dependent pressure and size threshold, are termed stable cavitation. If an acoustic pressure beyond the threshold is delivered, the
microbubbles will reach a critical upper size limit beyond which they are too unstable to sustain their structure as fluid rushes inward against their
growing shell. This causes the microbubble to collapse during the subsequent compression phase, leading to microjetting and bubble implosion.
This implosion releases substantial energy, including hot spots with temperatures 4000−25,000 K, the release of light (sonoluminescence) and
pressure shock waves >800 atm.17,23−28
enhance sonosensitizer delivery, increase cavitation events,
amplify ROS generation, and mediate synergistic combination
therapy.
targeted, minimally invasive, nonionizing cancer therapy
sparing off-target tissue through the use of otherwise benign
sensitizers. However, as PDT is inherently hindered by the
relatively shallow (<1 cm) penetration depth of light through
tissue, its effectiveness against deep-seated tumors in a
minimally invasive manner is limited. Capitalizing on the
deep tissue penetration of ultrasound, SDT has therefore
emerged as a promising alternative to PDT14 for the treatment
of deep-seated tumors currently beyond the purview of PDT,
while still retaining its advantages over traditional cancer
therapies.
On the basis of these putative therapeutic advantages, the
investigation of SDT has grown in recent years, with 46% of
research articles within the field being published in the last 5
years alone (note: obtained by searching for “sonodynamic
therapy” within keywords, abstract, or title in Scopus). To date,
although preclinical in vitro SDT studies have shown promising
results across a variety of cancer cell lines as reviewed by
Rosenthal et al.,15 few studies have shown significant
longitudinal tumor regression in vivo. And so, in spite of
their similarities, SDT has yet to gain the preclinical and
clinical success enjoyed by PDT.16 This may largely be
attributed to a lack of clarity and consensus surrounding the
mechanisms that underly SDT. In the face of such ambiguity
and dispute regarding mechanisms within the field, the
optimization required for SDT to reach its clinical potential
as a novel anti-neoplastic treatment is hindered. Motivated by
this limitation, we hope to critically overview the currently
proposed mechanisms of SDT and strategies that may advance
the field. To this end, we particularly focus on SDT strategies
that apply nanomedicines, which have recently become the
most widely explored sonosensitizers, encompassing 224 of the
300 SDT studies published in the last 5 years (obtained by
searching for “sonodynamic therapy” within keywords,
abstract, or title in Scopus). We highlight advantages
nanomedicines may offer in enhancing the anticancer
therapeutic efficacy of SDT, including their potential to
2. MECHANISMS GOVERNING SONODYNAMIC
THERAPY
SDT is founded in sonochemical and sonomechanical events
that are thought to exert cytotoxic effects through the
generation of ROS or mechanical stresses, respectively.
These events are the result of microbubble acoustic
cavitation17−19 and thus in order to overview the mechanisms
of SDT, it is first necessary to establish the types of acoustic
cavitation through which it may arise.
2.1. Acoustic Cavitation. As shown in Figure 1, acoustic
cavitation involves the formation, growth, and collapse of
bubbles in response to a pressure pulse. These bubbles, or
cavitation nuclei, can be exogenously delivered (this will be
discussed in a subsequent section), or as more prominently
explored in the SDT field, generated from a driving acoustic
pulse within biological fluids. These cavitation nuclei typically
are located at the boundary of a fluid and within gas filled
crevices,20 such as the cellular cytoplasm. The existence of
these nuclei has been extensively researched and confirmed,
while the mechanism behind their creation and stabilization
continue to be explored.21,22 Nevertheless, the field of SDT
predominantly assumes that cavitation leading to sonosensitizer activation arises from the de novo formation of
endogenous microbubbles in response to a driving acoustic
pressure wave.
Cavitation of these exogenous and endogenous bubbles can
be classified as either stable or inertial, each of which yields
different bioeffects. At a defined frequency, application of a
pressure pulse causes diffusion of gas into the bubble during
expansion and out of the bubble during contraction.29 During
stable cavitation, bubbles expand and contract around the same
resting radius in response to the acoustic field phases of
compression and rarefaction30 (Figure 1). At increasing
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reported in the form of power density output unless otherwise
noted. It is our hope that acoustic pressure will be standardly
reported in future SDT studies to better contextualize and
understand the bioeffects and proposed mechanical ROSindependent and ROS-dependent mechanisms of action
underlying SDT.
2.2. ROS-Based Mechanisms. Given its inspiration from
PDT, it is unsurprising that the predominant mechanism of
action proposed to instigate SDT cytotoxic effects is through
the formation of ROS. Similar to PDT, it is proposed that the
release of short-lived ROS causes oxidative damage in proteins,
lipids, and DNA.39 Several studies have identified ROS
generation following SDT in vitro and in vivo using ROS
quenchers,40 probes,41 and electron paramagnetic resonance
spin trapping42 across different sonosensitizers and ultrasound
parameters. In one such study, Umemura et al.18 observed cell
damage after applying the gallium-porphyrin analogue ATX-70
as a sonosensitizer and 4.5 W/cm2 ultrasound to sarcoma 180
(S180) cells. Introduction of 10 mM histidine, a singlet oxygen
quencher, was found to rescue cells and exude a sonoprotective
effect. Addition of mannitol, a ·OH quencher, did not display
such sonoprotection. As such, it was suggested that 1O2 is the
predominating ROS that causes SDT effects. Similar results
were observed by groups utilizing DCPH-P-Na,42 5-ALA,43
HAC,44 PpIX,45 Photofrin II,46 HpD,41 Rose Bengal,47
Erythrosin B,48 Piroxicam,49 and pheophorbide A50 as
sonosensitizers. However, ROS generation beyond 1O2 has
also been implicated in mediating SDT effects. For example,
the thermolysis of H2O to ·H and ·OH following inertial
cavitation-localized-heat generation has also been proposed to
be the source of SDT-triggered ROS generation that can either
kill nearby cells directly as primary free radicals or undergo free
radical oxidative transformation, the products of which are
sonosensitizer-dependent.51
Overall, several studies over the last three decades have
observed a variety of ROS being generated in vitro following
concurrent exposure of cells and solutions to low frequency
ultrasound and sonosensitizer52−57 (Figure 2a). Regardless of
the identity of ROS generated, the exploration of downstream
biological effects has reinforced the school of thought that
ROS-based mechanisms predominate SDT phenomena. Such
studies looking at ROS-mediated effects have thus far
implicated ROS to trigger loss in cell membrane integrity,
activation of the mitochondrial-apoptosis pathway58 and
oxidative DNA damage,59 ultimately causing cell death (Figure
2b,c).
For example, Tang et al. investigated membrane fluidity,
morphology, and enzyme activity in vitro in S180 cells posthematoporphyrin SDT treatment with 1.75 MHz, 1.4 W/cm2
continuous wave ultrasound. The authors observed increases in
lipid peroxidation and phosphatide degradation with a
corresponding decrease in membrane fluidity post-SDT when
compared to ultrasound alone, suggesting alterations in
membrane structure and lipid composition to play important
roles in mediating SDT effects.61 In further studies, cell
membrane damage was visually confirmed via SEM images of
HL-60 cells exposed to 255 kHz 0.4 W/cm2 ultrasound with or
without the administration of the sensitizer merocyanine 540.
Clear surface pores and extrusion of the cytoplasm were
identified in cells treated with SDT (Figure 2b(i-ii)) but not
ultrasound alone (Figure 2b(iv)), indicating cell membrane
porosity as a contributor to therapeutic effects.60 These
morphological differences between ultrasound alone versus
acoustic pressures, nonlinear bubble oscillations arise, creating
strong shear forces and viscous shear rates near the surface of
the bubble. This nonlinear stable cavitation generates
mechanical shearing and microstreaming in nearby environments,31 shown to produce stresses sufficient to disrupt cell
membranes.32 Depending on the degree of energy input, this
may in turn result in cell death through exposure of the
protoplasm to the cell’s external environment. On the basis of
their bioeffects, it is these nonlinear oscillations that will
henceforth be referred to as stable cavitation within this review.
With increasing ultrasound intensities above a given pressure
threshold, bubble expansion occurs beyond a given resonant
size, leading to a loss of stability. This yields inertial cavitation,
whereby inward rushing of fluid against the unstable,
expanding bubble causes microjetting, bubble collapse, and
implosion (Figure 1). This results in energy release in the form
of a shockwave yielding transient (350 ps to <2 μs for a single
bubble33) sonochemical hotspots corresponding to a liquid
layer roughly 500 molecules thick, modeled temperatures
ranging 4000−25,000 K23−25 and pressures above 800
atm.17,26,27 This release of thermal energy may trigger
sonochemical reactions generating ROS and sonoluminescence, while the resultant shock waves are capable of
mechanically disrupting nearby cell membranes.32,34 As such,
while it is anticipated that any SDT therapeutic effects
generated in the realm of stable cavitation may be a result of
mechanical forces, bioeffects from inertial cavitation may be
both sonochemical and sonomechanical in nature. It is thus
important to differentiate SDT from the predominantly
mechanical effects of thermal and nonthermal HIFU. HIFU
thermal ablation makes use of high ultrasound intensities to
locally elevate tissue temperatures and cause tissue necrosis.3
Conversely, SDT effects have either shown to be, or are
assumed to be, nonthermal or minimally hyperthermic in
nature.35,36 Nonthermal HIFU ablation in the form of
histotripsy arises from inertial cavitation, but makes use of
shorter ultrasound pulses (on the order of μs) and higher
ultrasound intensities (exceeding 10 MPa and 1000 W/cm2 for
histotripsy versus <10 MPa and <10 W/cm2 for SDT when
reported) than typically used in SDT, leading to mechanical
nonthermal homogenization of tissue targets.4,37,38 Ultimately,
SDT requires the use of a sonosensitizer to impart therapeutic
effects that are not solely sonomechanical in nature, making it
fundamentally different from HIFU thermal ablation or
histotripsy, which does not require any sonosensitizer
administration.
Consequently, in order to differentiate between stable/
inertial cavitation and between HIFU/SDT effects, reporting
of acoustic pressure, beam characteristics, target tissue
histology, and detection of cavitation are required. Unfortunately, as will be more thoroughly discussed in later sections,
there is a lack of universal reporting of the above parameters
within the field of SDT. Field parameters are highly dependent
on the ultrasound setup. Given the diverse range of ultrasound
apparatus, this absence of apparatus calibration and pressure
reporting generates uncertainty when drawing comparisons
between SDT studies or when drawing generalized conclusions
from any observed biological effects. While we do attempt to
differentiate SDT mechanisms and bioeffects based on
cavitation phenomena that are pressure dependent, most
studies within the field opt to present acoustic intensity in the
form of device-dependent power density rather than calibrated
pressure measurements. As such, ultrasound parameters will be
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Figure 2. continued
sensitizer or ultrasound-only controls. Evidence of cell membrane
damage was observed by Tachibana et al. when comparing SEM
images of HL-60 cells exposed to SDT (i,ii; merocyanine +255 kHz
0.4 mW/cm2 ultrasound) versus those of untreated cells (iii) or cells
exposed solely to ultrasound (iv).60 The formation of cell membrane
pores has been postulated to be a result of lipid peroxidation from
ROS generated post SDT. ROS generation has also been associated
with the induction of apoptotic cell death through mitochondrial
membrane potential disruption, activation of the Fas death receptor
pathway, or changes to the structure of DNA as summarized in (c).
Scale bar = 5 μm. The graphic in part (a) was adapted from Cheng et
al.54 (Copyright Dove Medical Press Ltd.) and part (b) with
permission from Tachibana et al.60 Copyright 1999 Elsevier.
SDT combined with the higher degree of lipid peroxidation
observed post-SDT in the literature suggest that SDT results in
cell membrane damage beyond that of cavitation-mediated
effects alone. These observed differences are consistent with
previously known ROS-based lipid peroxidation mechanisms,
including the ability of 1O2 to react with polyunsaturated fatty
acids in the membrane to cause peroxidation of membrane
lipids through the lipid hydroperoxide chain reaction.62,63
Therapeutically, this may yield apoptotic pathways similar to
other membrane destabilizing cytotoxic agents64 such as
potential inactivation of ion channels,65 transport proteins,66
or activation of the Fas death receptor pathway.
In addition to loss of cell membrane integrity, real-time loss
of mitochondrial membrane potential (MMP) and activation
of p53 tumor suppressive pathways have also been associated
with SDT. Honda et al. observed a monotonic increase in
MMP with increasing ultrasound intensity applied to U937
myelomonocytic lymphoma cells.67 Disruption of MMP has
been further implicated in ultrasound-induced apoptosis owing
to well-known capabilities of the mitochondria to induce the
self-destruct mechanism through caspase activation. Particularly, the disruption of electrochemical gradients observed
may be extrapolated to known mechanisms of cristae
organization disruption and inhibition of mitochondrial fusion
to inhibit function and therefore compromise cellular energy
supply.68 Follow-up studies investigating downstream events in
conjunction with ROS generation revealed implication of
nuclear factor-ΚB and tumor suppressor gene p53 in cellular
apoptosis following SDT. Specifically, selective transactivation
of p53 target genes was observed following Hp-mediated
SDT.69 Later experiments further confirmed activation of the
p53/caspase-3 apoptosis axis following sinoporphyrin sodium
(DVDMS)-mediated SDT.70 Hyper-physiological levels of p53
are known to influence cellular redox by activating proapoptotic factors PUMA, Bax, and Fas through transcriptiondependent and independent pathways.69 p53 is central in
mediating and integrating cellular response to DNA damage,
growth factor withdrawal, and oncogenic transformation,71
although these results do appear to be dependent on cell type
in SDT. Regardless, ROS-mediated effects on p53 creates
intriguing possibilities for combination therapy with SDT given
the role of p53 modulation in enhancing combinatorial
therapeutic efficacy with chemotherapeutics and radiotherapy.72 This highlights the importance of characterizing the
biological effects of SDT, as knowledge of specific downstream
pathways may allow for rational sonosensitizer and combination therapy design.
Figure 2. Biological effects of SDT are thought to be predominantly
caused by ROS generation (a) followed by a loss in cell membrane
integrity (b). Cheng et al.54 demonstrated ROS generation in THP-1
macrophages following the administration of 5-ALA-mediated SDT
(1 MHz, 10% duty cycle, 100 Hz pulse repetition frequency, 0.5 W/
cm2) using DCFH-DA, an intracellular fluorescent ROS probe.
Quantification of fluorescence (n = 6) demonstrated significant
intracellular increases in ROS generation following SDT compared to
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Figure 3. ROS-dependent SDT mechanisms initiated by inertial cavitation. The resulting sonoluminescence and heat (4000−25,000 K) activate
nearby sonosensitizers (shown in green) through (a) a PDT-like mechanism or (b) pyrolysis. Sonoluminescent light is thought to excite
photoactive sonosensitizers from the ground state to a short-lived excited singlet state (a−i). Following intersystem crossing, the resulting triplet
state can interact with cell substrates and oxygen to generate free radicals through a Type I PDT reaction or can directly interact with molecular
oxygen to generate cytotoxic singlet oxygen (1O2) via a Type II PDT reaction. This is made feasible through the emission of sonoluminescent light
over a broad range encompassing the Soret band of many porphyrin sonosensitizers as demonstrated by Umemura et al. (a-ii,iii).76 When
sonicating solutions (1.93 MHz, 0.6 MPa) containing hematoporphyrin (Hp), a decrease in the intensity of this sonoluminescent light was
observed at the Soret band of Hp without any observation of corresponding porphyrin fluorescence at wavelengths >600 nm, suggesting absorption
of light by the Hp (a-ii). When Hp and 1.92 MHz, 1.8 W/cm2 ultrasound were administered to Sarcoma-180 cells, decreases in viability were
observed that were only discernibly recovered with the addition of the 1O2 scavenger histidine, but not the OH· scavenger mannitol, supporting the
proposed involvement of sonoluminescence-mediated Type II sensitizer activation (a-iii). Alternatively, pyrolysis of sonosensitizers may be the
source of ROS generation following SDT. The high focal temperatures generated from inertial cavitation can cause the thermolysis of water,
yielding free radicals that can interact with sonosensitizers to generate longer-lived cytotoxic peroxyl radicals. Sonosensitizers may also be directly
decomposed into radical species in the vicinity of the sonochemical hotspot. The formation of these sensitizer-derived peroxyl radicals was observed
by Mišiḱ et al.,77 who observed increasing peroxyl radical generation with increasing sensitizer concentration in solution (ii) that correlated with a
decreasing concentration of ·OH. Adapted with permission from Umemura et al.78 (Copyright 1990 John Wiley and Sons) and Mišiḱ et al.77
(Copyright 1996 Elsevier).
sonosensitizer activation and greater ultrasound/sonosensitizer
synergy in the absence of confounding effects from inertial
cavitation.
Once generated, it is stipulated that sonoluminescence may
stimulate a photochemical reaction similar to that of PDT.
Here, the sonoluminescent light may excite sonosensitizers to a
short-lived singlet state Sn (Figure 3a(i)). Following intersystem crossing to a triplet state, the sonosensitizer may then
interact with biological substrates to generate ROS similarly to
a Type I PDT pathway, or by directly interacting with
molecular oxygen to generate cytotoxic singlet oxygen (1O2)
analogous to Type II PDT. Due to its resemblance to PDT, the
sonoluminescent pathway of SDT ROS generation has
directed the use of photosensitizers as sonosensitizers. To
this end, porphyrins have most widely been explored as
sonosensitizers given their ability to undergo photochemical
processes to generate ROS via type I or type II PDT.79
Early work from Umemura et al. thus employed porphyrin
sonosensitizers to shed light on both the origin and the utility
of sonoluminescence in SDT. In initial studies using
hematoporphyrin (Hp) as a sonosensitizer,76 the authors
observed the emission of visible light between 400 and 450 nm
when applying 600 kPa, 1.93 MHz ultrasound to a solution of
saline (Figure 3a(ii)). This broad emission was also observed
Should a ROS-based mechanism be implicated in SDT, the
natural course of action would be to maximize ROS yields in
future sonosensitizer design. In order to do so, it is first
important to understand the pathways by which sonosensitizers in the vicinity of cavitating bubbles mediate ROS
generation. Currently, there are two primary proposed
cavitation phenomena that are thought to activate ROS
generation: sonoluminescence and pyrolysis (Figure 3).
2.2.1. Sonoluminescence. Sonoluminescence, first observed
in 1934 by Frenzel et al.,28 describes the phenomenon through
which the energy released from the rapid collapse of a bubble
during acoustic cavitation instigates a very brief emission of
light. While generally associated with inertial cavitation due to
the rapid release of energy resulting from bubble implosion,
two studies have reportedly observed sonoluminescence at
acoustic pressures and amplitudes attributed to stable
cavitation.73,74 Given the lack of extreme energy output in
stable cavitation, theoretical models have suggested that the
gaseous phase within the bubble can attain Tmax similar to
inertial cavitation 75 to generate photons and thereby
sonoluminescence via stable cavitation.73 Although minimal
study has gone into analyzing the possibilities of such an event,
the generation of sonoluminescence under conditions of stable
cavitation would promisingly allow for more control over
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would combine such sonoluminescence detection techniques
(including the in vitro use of photomultiplier tubes, charged
couple device surface imaging, or focal ultrasound tomography) with cavitation detection and pharmacokinetically
similar photoinactive and photoactive sonosensitizers when
exploring ROS generation and associated therapeutic effects
from SDT. Furthermore, sonoluminescence detection within
mammalian tissue, which remains to be a challenge, may be an
avenue that could allow clearer mechanistic conclusions to be
derived in vivo.
2.2.2. Pyrolysis. In lieu of sonoluminescence, an opposing
theory for ROS-mediated SDT has arisen that postulates
sonochemical pyrolysis following inertial cavitation to cause
SDT effects. Under this mechanism, cavitation nuclei act as a
combustion-chemistry reactor after the extreme energy release
from the collapsing cavity. Accordingly, the high local
temperatures and pressures generated from inertial cavitation
may directly lead to pyrolysis of the bubble components or of
the solvent vapor.86 Assuming an adiabatic model of collapse of
a spherical bubble, the heat conductivity from the final
temperature at the moment of collapse (4000−25,000
K)23−25,34 into the liquid may be sufficient to cause pyrolysis
of molecules at the gas−liquid interface to generate free
radicals.87 This proposition was supported by Riesz et al.34
who detected the generation of ·OH and ·H following the
sonication of aqueous solutions at 1 MHz and 2 W/cm2
intensity ultrasound. Assuming that recombination or disproportionation does not occur, these generated free radicals
could act indiscriminately against macromolecules to induce
cytotoxic damage.
While many studies investigating induction of SDT
cytotoxicity through pyrolysis are conducted independent of
a sonosensitizer, it is hypothesized that the activation of a drug
in proximity to the collapsing sonochemical reaction can
amplify the effects generated by ultrasound alone. With the
presence of a sonosensitizer, either a lowered inertial cavitation
pressure threshold or increased generation of ROS are thought
to cause the synergistic effects observed within SDT. However,
there remains doubt regarding the contribution of intracellularly generated ·OH following inertial cavitation. Studies
have theorized immediate cell destruction in response to
intracellular cavitation caused by an oscillating bubble of a size
far beyond that of a typical cell.88,89 In this event, the short
half-life (∼1 μs for 1O2, ∼1 ns for ·OH),90 high reactivity, and
limited diffusion distances of ROS (∼20 nm for 1O2, ∼5 nm
for ·OH)91 may limit their involvement in imparting
cytotoxicity following inertial cavitation. Assuming extracellular
cavitation, there may exist limitations by which ROS can react
with critical cellular sites such that only those proximal to the
area of production are affected.92 Based on this, it has been
proposed that pyrolysis of sonosensitizers may create free
radical intermediates beyond ·OH and H·, such as peroxyl
radicals. These less reactive, longer-living radicals may then, in
theory, be able to migrate the necessary distances to cell
membranes where lipid peroxidation and cell death would
occur.93 Various studies contradicting this postulate typically
revolve around the protective effect of cysteamine, a cell
membrane-permeable free radical scavenger, and the lack of
protection provided by cystamine, a membrane-impermeable
free radical scavenger94,95 against SDT. Further information
contending this debate can be found in extensive detail in
reviews by Church et al.96,97 and Miller et al.88
with the addition of ROS scavengers, specifically those acting
on 1O2, OH·, and O2−, suggesting that sonoluminescence was a
result of cavitation hot spots rather than from the
recombination of ROS. Noting the overlap between the peak
absorbance of Hp at 411 nm80 and the broad sonoluminescence peak between 400 and 450 nm, the authors hypothesized
activation of the sonosensitizer either through nonequilibrium
energy transfer from sonochemical hotspots or by direct
photoactivation from the generated light. On the basis of this
study, multiple papers have investigated sonoluminescence as a
mechanism of SDT action in vitro and in vivo.41,81,82 Umemura
et al.’s seminal work also illustrates the requirements needed
for a PDT-like sonoluminescence pathway of SDT action to
ensue: (1) a sensitizer with a high yield of intersystem crossing
to generate an excited triplet state, and (2) overlap between
the peak absorbance of the sensitizer and the broad emission of
sonoluminescence. It should also be noted that only through
the triplet state can energy be transferred to nearby O2
molecules to generate 1O2. Thus, the observation of 1O2
generation in SDT studies is often associated with the
presumption that sonoluminescence is the source of sensitizer
activation.
There have, however, been studies that contradict
sonoluminescence-mediated photodynamic generation of
ROS as the SDT mechanism of action. Of particular interest
was a study that made use of 13,17-bis(1-carboxyethyl)-8-[2(2,4-dichlorophenyl-hydrazono)ethylidene]-3-ethenyl-7-hydroxy-2,7,12,18-tetramethylchlorin, disodium salt (DPCH-PNa), an agent claimed to be photoinactive.83 Despite presumed
photoinactivity, Hachimine et al. observed statistically
significant sonotoxicity in vitro and in vivo when compared
to ultrasound alone at 1 MHz, 2 W/cm2, contradicting the
belief that SDT activity is based on sonoluminescent
photoactivation of sensitizers. Sonotoxicity on cell viability
was blocked and reached ultrasound-alone levels with the
addition of histidine but not mannitol, suggesting the
involvement of 1O2. However, although claimed to be
photoinactive, DPCH-P-Na did cause statistically significant
cell toxicity when compared to controls upon photoirradiation
with 60,000 lx for 10 min, making it difficult to exclude
sonoluminescence completely from a mechanistic perspective.
Nevertheless, other studies have also demonstrated the
generation of ROS from sensitizers that would typically be
photoinactive. For example, copper protoporphyrin (Cu-PP)
was found to produce cytotoxic effects in response to albumin
microbubble-mediated SDT using 3.2−4.0 W/cm2 acoustic
intensity spatial peak temporal average on L1210 cells.84 This
result suggested that sonoluminescence was not responsible for
the cytotoxic effects observed, as the short triplet state lifetime
of the metalloporphyrin would yield poor generation of 1O2
upon photoexcitation. Other compounds, such as piroxicam,
have also been explored to induce cytotoxic effects from 1O2
generation49 despite their low singlet oxygen yield.85
Collectively, these studies suggest that SDT is governed by
mechanisms of action beyond simple sonoluminescencemediated PDT-like photoactivation of sonosensitizers.
Indeed, the exclusive observation of efficacy resulting from
porphyrin-derived sonosensitizers is insufficient as conclusive
evidence for sonoluminescent SDT activation. Further
comprehensive studies with clinically relevant ultrasound
parameters, light controls, sonoluminescent detection techniques, and ROS probes with high specificity are required to
investigate this possibility. Ideally, these mechanistic studies
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To this end, water-based azo compounds have been
explored as sonosensitizers due to their ability to decompose
to carbon-centered alkoxyl radicals and ultimately peroxyl
radicals in the presence of oxygen.52 Both radical species were
successfully identified after sonication through spin trapping
and electron paramagnetic resonance spectroscopy, indicating
successful decomposition of the sonosensitizer. Indeed,
quenching with potassium iodide, sodium azide, and sodium
formate did not decrease the concentration of radicals present,
suggesting a thermolysis/pyrolysis-mediated effect of the
sonosensitizer as opposed to an 1O2 or ·OH-mediated
response. Crucially, however, these experiments were conducted in vitro at low frequency ultrasound (50 kHz) in a
standing wave field, whereupon efficacy is often directly related
to a given spatial average, temporal average ultrasound
intensity.98 Variations in geometry between ultrasound systems
make it difficult to compare cavitation or estimate acoustic
intensity delivered. Standing wave production is also known to
promote cavitation by virtue of bubbles below resonant size
collecting at pressure maximums.99 Collectively, this nonstandard ultrasound delivery prevents the observation of
peroxyl-mediated cytotoxicity observed in this study to be
extended broadly, necessitating further studies to explore the
mechanism of decomposition of azo compounds using
translatable ultrasound parameters. As a whole, pyrolysis
within SDT is not well understood and the products produced
from different classes of sonosensitizers remain to be
elucidated. Mechanistic studies that make use of sonosensitizers that can amplify pyrolysis may thus be of interest.
2.3. ROS-Independent Mechanisms. While ROS-mediated effects are generally accepted as the dominant mechanism
underlying SDT, one must account for the inherent
sonomechanical effects induced by ultrasound. Ultrasound
irradiation alone can hydrodynamically shear cells through
acoustic microstreaming, pressure pulse-mediated erosion, and
microjetting, all of which can ultimately cause cell membrane
disruption.100−102 Initial work conducted by Worthington et
al.103,104 supported sonomechanical events leading to SDT
effects as opposed to ROS generation. The authors
investigated HO· and H· yield through Fricke dosimetry
following 1.955 MHz variable intensity ultrasound (where 1.2
W/cm2 ≈ 0.19 MPa) applied to both water and PBS, with or
without hematoporphyrin (Hp), and shaken or tilted to
stimulate microbubble formation. No singlet oxygen luminescence could be measured with or without Hp post-36 W/cm2
ultrasound despite confirmation of cavitation signal, notable
cell death, and detectable luminescence using corresponding
PDT treatment. Given the intensities used, it comes as little
surprise that sonomechanical damage was interpreted to be the
predominant mechanism of cell death given the known
associated cavitation and potential thermal effects. While this
study fails to incorporate any clinically relevant ultrasound
parameters in its SDT studies, it may loosely be taken to
provide opposition toward a ROS-based theory that has yet to
be fully addressed.
Although the mechanical effects of acoustic cavitation alone
have been widely studied, it remains unclear as to how, or if,
the addition of a sonosensitizer will mediate a synergistic
cytolytic response. Current theories propose that the addition
of a membrane destabilizing compound can enhance physical
stresses induced by ultrasound to mediate a synergistic effect
(Figure 4). While few studies have looked in depth at
sonomechanical effects in the presence of a sensitizer, previous
Review
Figure 4. ROS-independent mechanisms of SDT actions. It is thought
that sonosensitizer incorporation within cell membranes causes their
destabilization. This in turn is thought to make the cell membrane
more vulnerable to cavitation mechanical events such as microjetting,
microstreaming, and shock waves resulting from inertial cavitation.
Thus, sonosensitizers may enact their synergistic SDT effects by
lowering the pressure threshold needed for sonomechanical cell
damage and subsequent death.
research analyzing acute mechanically induced hemolysis
suggests a role of drugs, in this case, Vitamin E, in making
cells more vulnerable to lysis.105 Follow-up studies employing
Trolox, a derivative of Vitamin E, demonstrated that the drug
enhanced shear forces following ultrasound-induced hemolysis.106 This enhanced membrane-destabilization via drug
agents may in turn be applied to SDT. It will therefore be of
interest to characterize the interactions between sonosensitizers and cell membranes to elucidate how, or if, this
interaction amplifies cavitational effects. Porphyrins in
particular have previously been modeled to interact with cell
membranes through several types of forces: electrostatic,
hydrophobic, hydration, and coordination. The hydrophobic
porphyrin core is known to embed into the lipid region of cell
membrane bilayers, creating not only preferential accumulation
of the molecule, but also potential enhancement of mechanical
stresses by destabilizing the membrane.107 It is important to
note that while these theorized porphyrin−membrane
interactions may lead to enhanced sonomechanical effects,
they are thus far based on photosensitizer-membrane modeling
performed for PDT and not SDT. Given the well-known role
of sonomechanical damage in ultrasound, it is worth exploring
its impact within SDT across the different sonosensitizers in
use. Furthermore, exploration of such presumed sonomechanical events within the realms of both stable and inertial
cavitation may lead to better clarification of the contribution of
mechanical versus ROS-mediated effects. For example, when
applying stable cavitation from ultrasound alone to U937 cells,
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Table 1. Summary of Proposed Mechanisms Underlying the Effects of SDT
Mechanism
Supporting Evidence
Contradicting Evidence
Unknowns
Sonoluminescence
Overlap observed between peak absorbance of porphyrin sonosensitizers and sonoluminescence peak
Triplet state to generate 1O2 −
observed through electron paramagnetic resonance spin trapping,
probes, quenchers
·OH, H·, long-lived peroxyl and
alkoxyl species detected via electron
paramagnetic resonance spin trapping, probes, quenchers, indicating
decomposition of sonosensitizer
Short half-life of ROS limits migration
within cellular sitesmust require
longer-lived radicals to mediate
observed therapeutic response
Known ability of ultrasound to cause
physical damage
Membrane destabilizing compounds
enhance ultrasound-induced hemolysis
Metalloporphyrin and other compounds with low
singlet oxygen yield (ex, piroxicam) yield
cytotoxic and therapeutic efficacy despite short
triplet state lifetime
Intensity, power, duration,
wavelength of light emitted
from sonoluminescence
Characterization of light
emitted in relation to the
class of and structure of
sonosensitizer used
Products produced from different classes of sonosensitizers can vary greatly in
both structure as well as
mechanism of pyrolysis
41,76,81,82
Impossible to isolate physical
stressors from the generation of ROS during inertial cavitation
88,101,102,105,106,108
Pyrolysis
Mechanical
Protective effect observed by cysteamine, a cell
membrane-permeable free radical scavenger,
and lack thereof by cystamine, a membraneimpermeable scavenger, suggesting activity of
ROS within the cellular membrane
Generation of ROS in most studies likely
indicates this is not the sole mechanism at play
Refs
34,52,88,91,94−97
comprehensively explore the contribution of sonoluminescence, pyrolysis, and mechanical effects in vitro and in vivo
across different classes of sonosensitizers and cavitation
paradigms (stable and inertial) is warranted. This will allow
for better elucidation of sensitizer-specific mechanisms of SDT
and the derivation of structure−activity relationships for
optimal sonosensitizer development and ultrasound parameter
selection.
Feril et al. showed neither free radical detection nor oxidative
stress response despite inducing 70.0 ± 13.8% apoptosis in
vitro.108 Increasing the applied acoustic pressure was associated
with both free radical detection and oxidative stress response,
as well as further decreases in cell viability and increases in
secondary necrosis, showcasing the combinatorial sonomechanical and sonochemical effect known to be a part of inertial
cavitation. Since it is impossible to isolate physical stressors
from the generation of ROS during inertial cavitation, studies
focusing on SDT under parameters associated with stable
cavitation may shed light on the mechanical contributions of
acoustic cavitation in SDT.
2.4. Summary of Mechanisms and Future Outlook.
The driving force behind SDT is the interaction of
sonosensitizers with cavitating bubbles in biological media
through ROS-dependent and independent mechanisms, as
summarized in Table 1. Acoustic cavitation, specifically inertial
cavitation, can generate hot spots giving rise to high
temperatures and sonoluminescence that can in turn trigger
the generation of sonosensitizer-derived ROS, although stable
cavitation could also be implicated in ROS generation.
Sonosensitization may alternatively or additionally be a result
of drug-induced membrane destabilization, lowering the
threshold for cell membrane damage by sheer forces generated
from acoustic cavitation. However, studies thus far exploring
the therapeutic utility and potential mechanisms at play in
SDT largely indicate that therapeutic effects, particularly in
vitro, are mediated through ROS. This overarching basis of
SDT was also supported in vivo by McEwean et al., who
demonstrated that oxygen-containing microbubbles suppressed
tumor growth to a greater extent than equivalent microbubbles
loaded with sulfahexafluoride gas following Rose Bengalmediated SDT in BxPC3 subcutaneous tumor-bearing mice.109
The observation of oxygen-amplified therapeutic effects
supports a ROS-mediated therapeutic mechanism of action,
which would require the presence of molecular oxygen.
Nevertheless, the overarching pathways and patterns leading
to such ROS generation remain unclear. Given the differences
in physiochemical properties across sonosensitizers, it is logical
to assume that each class follows different means of acoustic
activation with differing degrees of contributions from each of
the mechanisms proposed. Thus, mechanistic studies that
3. SONOSENSITIZERS
Thus far, we have briefly overviewed how SDT is instigated in
the context of acoustic cavitation. Understanding such
mechanisms may allow for more purposeful sensitizer selection
and thereby improved therapeutic outcomes. Advances in
sonosensitizer chemistry in parallel are also imperative for
congruently establishing greater SDT efficacy. Traditionally,
SDT has made use of molecular sonosensitizers. Multiple
comprehensive literature reviews have detailed the diverse
range of molecular sonosensitizers used preclinically110−113
and thus will not be covered in depth within this review. Thus
far, numerous classes of molecular sonosensitizers have been
used in literature including porphyrins,76 xanthene dyes,47
chemotherapeutic anthracycline derivatives,114 azo-compounds,52 heptamethine dyes,115 nonsteroidal anti-inflammatory drugs (NSAIDs),49 and many more. However, it is
debatable whether the observed increases in cytotoxicity in
some of these drug classes were due to sonodynamic activation
of these drugs as would be expected for SDT, or merely a result
of increased drug delivery. Accordingly, sonosensitizers
without inherent toxicity have been primarily researched to
better differentiate SDT synergistic effects from those imparted
by the drug itself. Particularly, porphyrin-based photosensitizers are of great focus owing to their propensity to produce
ROS upon photoactivation. Their ubiquitous use, however,
places a greater emphasis on the role of sonoluminescence in
SDT than would be with the use of photoinactive
sonosensitizers. As such, there remain many avenues for
sonosensitizer innovation that may enhance SDT efficacy and
translation. To this end, supramolecular sonosensitizer
assemblies, particularly in the form nanoparticles, have recently
advanced the therapeutic efficacy and feasibility of SDT as a
cancer treatment strategy as will be discussed herein.
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Figure 5. Enhancing SDT through nanomedicines. Encapsulation of sonosensitizers within nanoparticles can improve bioavailability and increase
drug delivery to cancer cells by increasing drug solubility, facilitating accumulation via the enhanced permeability and retention effect, cell-specific
targeting and on-demand ultrasound-triggered sensitizer release (a). This was exemplified by Zhang et al.129 who demonstrated that encapsulation
of HMME within liposomes allowed for ultrasound-mediated drug release (i), higher tumor accumulation of liposomal ICG versus free ICG (ii),
and ultimately delayed tumor growth of liposomal HMME following the administration of SDT to MCF-7 tumor-bearing mice (iii) (adapted with
permission from ref 129, Copyright 2019 John Wiley and Sons). Nanoparticle formulation of sonosensitizers also holds the advantage of providing
alternative pathways to sonosensitization (b). This can include the use of inorganic nanoparticles, such as (i) TiO2 as a new class of ROSgenerating sonosensitizers (adapted with permission from Harada et al.,131 Copyright 2013 Royal Society of Chemistry), (ii) increasing
microbubble cavitation in solutions (adapted with permission from Tuziuti et al.,142 Copyright 2005 American Chemical Society), and (iii) creating
more cavitation nuclei in solutions containing nanoparticles (bottom two panels), versus solutions devoid of nanoparticles (top two panels; adapted
with permission from Pan et al.,143 Copyright 2018 John Wiley and Sons) to enable improved therapeutic efficacy. ROS generation can also be
amplified by nanomedicines through the codelivery of sonosensitizers with oxygen to overcome tumor hypoxia, coloading with agents activated by
SDT-induced hypoxia to generate ROS and reducing intratumoral GSH levels to prevent scavenging of ROS generated by SDT (c). Coloading of
oxygen with the sonosensitizer IR780 increased ROS generation intracellularly (i), which translated into increased survival compared to SDT
conducted in the absence of oxygen loading (ii) (adapted with permission from Chen et al.,144 Copyright 2017 American Chemical Society).
Conversely, SDT itself can result in consumption of oxygen and tumor hypoxia (iii) as evidenced by observable HIF-1α staining of PC3 tumor
slices following the application of SDT in comparison to control tumors.145 This tumor hypoxia was exploited to activate the hypoxic prodrug TMZ
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Figure 5. continued
to enable bioreductive therapy (BRT) that delayed tumor growth compared to SDT alone (iv) (adapted with permission from Wang et al.145
Copyright 2018 John Wiley and Sons). Finally, coencapsulation of multiple therapies within a single nanomedicine agent lends itself to spatially
colocalized combination treatments and synergistic therapy, including chemo/SDT, PDT/SDT, radiotherapy/SDT, immune/SDT, which can
overcome drug resistance and prevent tumor metastasis (d). The immunomodulatory abilities of SDT combined with TLR7 agonist therapy from a
single nanoparticle agent was demonstrated by Yue et al.146 The combination therapy increased dendritic cell maturation (i) and the release of
cytokines such as TNF-α (ii), which, when combined with anti-PD-L1 therapy, decreased primary and distant tumor growth (iii-iv) (adapted with
permission from ref 146, Copyright 2019 Springer Nature).
3.1. Role of Ultrasound Contrast Agents. Exogenously
delivered microbubbles have served as supramolecular
diagnostic agents for ultrasound imaging for decades.
Approved clinically to visualize tissue and cavity perfusion,
ultrasound contrast agents are micron-sized (1−8 μm
diameter) gas-filled microspheres surrounded by a lipid,
polymer, or protein shell in colloidal suspensions.116 As their
acoustic behavior is similar to that of endogenously formed
bubbles, it is proposed that microbubbles can lower the
threshold for SDT events by acting as preformed cavitation
nuclei,117,118 ultimately yielding higher therapeutic efficacy at
equivalent or lower acoustic intensities than agent-free
platforms.119 Preformed microbubbles, similar to de novo
bubbles, also have the ability to generate sonoluminescence at
intensities associated with inertial cavitation.120 Inclusion of
sonosensitizers within the microbubble shell can further
increase SDT-mediated cytotoxicity in a bubble-dependent
manner. For example, in examining the effects of Rose Bengal
(RB) either covalently attached to lipid microbubbles or
independently administered as a free drug agent alongside lipid
microbubbles under ultrasound irradiation (1 MHz frequency
and 1.5 W/cm2 power density for 60 min), Nomikou et al.
observed that the RB-microbubble conjugate increased cell
death in vitro compared to the unconjugated RB + microbubbles and sonosensitizer-alone SDT treatment groups.119 In
vivo studies were attempted using more therapeutically relevant
ultrasound parameters (1 MHz frequency and 3.5 W/cm2
power density for 3 min) showing no increase in LNCaP-Luc
tumor growth with RB-microbubble conjugate SDT after 12
days, although an in vivo comparison to unconjugated RB was
not conducted.119 Similar promising results have been
observed with microbubble-conjugated chemo-sonodynamic
therapy,121,122 as well as oxygen/RB-loaded microbubbles for
hypoxic tumor treatment.123 Thus, sonosensitizer-loaded
microbubbles may be a promising step forward toward
advancing the therapeutic effects of SDT, provided that
ultrasound dosimetry is clearly evaluated in a standardized
manner (for example, through acoustic pressure, and with
comparison against effects observed for free sensitizer and
microbubble-devoid control groups).
3.2. Role of Nanomedicine in SDT. While microbubble
agents amplify SDT effects by acting on acoustic cavitation,
nanoparticles deliver advantages more pertinent to sonosensitizer availability and activity. As such, nanoparticles account for
the most widely explored supramolecular variant of sonosensitizers in the literature. More specifically, nanosensitizers can
increase SDT efficacy by better facilitating sonosensitizer
delivery, generating new means of ROS generation, overcoming hypoxia, and allowing for spatially localized combination therapies to be explored (Figure 5; Table 2).
3.2.1. Drug Delivery. Recent decades have witnessed an
abundance of preclinical exploration of nanosized materials to
change the pharmacokinetics of free drug agents and maximize
their on-target delivery. Numerous studies using liposomal,
micellar, and polymeric nanoparticles within the scope of
chemotherapy drug delivery have shown cell targeting, delivery
across biological barriers, reduction in toxic side effects, and
avoidance of multidrug resistance protein 1 (MDRP1).124,125
Similar delivery advantages can be extended to nanoformulations of sonosensitizers. The hydrophobic nature of
many traditional organic sonosensitizers, such as porphyrin,
can reduce their bioavailability and, accordingly, therapeutic
concentration in vivo. Assembling these free sensitizers within
nanostructures can potentially overcome this challenge,
allowing for higher drug doses to reach tumor sites.126
Furthermore, the dense packing of sonosensitizer within a
single nanoparticle could allow for greater therapeutic efficacy
given the need for spatiotemporal localization of ultrasound
and sensitizers for effective SDT. Consequently, nanoparticle
systems may facilitate more potent SDT by improving
sonosensitizer delivery to target sites.
To this end, hollow mesoporous organosilica nanoparticles
(HMONs) have been explored as nanocarriers due to their
large surface area and pore volume.127 These traits can
respectively allow for dense covalent anchoring of sonosensitizers (PpIX) to maximize drug delivery, and for diffusion of
SDT-generated ROS out of the mesopores. When exposed to
PpIX-HMON SDT (1.0 MHz, 1.5 W/cm2, 2 min) in vitro, a
reduction of more than 70% cell viability was observed in 4T1
cells, while the nanoparticle itself showed no cytotoxicity.
When applied in vivo at 2.3 W/cm2, statistically significant
tumor growth inhibition was achieved over ultrasound alone.
In another drug loading study conducted by Liu et al.,128
sinoporphyrin sodium (DVDMS) was loaded into homotypic
tumor cell-derived exosomes. The authors observed superior
tumor suppression and ROS generation compared to both free
DVDMS as well as nonirradiated exosomes in a reportedly
synergistic manner. Delivery was also enhanced when the
nanoformulation of DVDMS was combined with ultrasound
treatment. Intracellular localization was observed to shift from
the lysosome to the mitochondria following US stimulation,
suggesting promotion of cargo transport and endosomal
opening to trigger cargo release. Enhanced tumor delivery
and penetration in vivo were further observed with ultrasound
versus without. This phenomenon of cavitation-mediated drug
release was further observed by Zhang et al.129 The authors
demonstrated that hematoporphyrin monomethyl ether
(HMME)-loaded liposomes could rapidly release 60% of the
loaded HMME when exposed to 1 MHz, 0.5 W/cm 2
ultrasound (Figure 5a). This translated to higher accumulation
of loaded imaging agents in MCF-7 tumors exposed to
ultrasound when compared to free agents, and ultimately
slightly slower tumor growth with the use of liposomal versus
free HMME. Thus, nanoformulations of sensitizers may
facilitate increased drug delivery to target lesions, possibly by
facilitating triggered drug release.
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Table 2. Summary of Nanomedicines Explored for SDT
Nanoparticle
HMONs
Sensitizer
PpIX
Function/Effect
Drug Delivery
Protected sonosensitizer from physiological environment,
enhanced tumor accumulation, sustained release
US
Frequency
1 MHz
DVDMS
Intensity/
Pressure
Ref
1.5−3.3
W/cm2
2−3 W
Pressures
unknown
127
128
Liposomes
Pyropheophorbide
Triggered delivery of coloaded sonosensitizer
1 MHz
0.2−0.3
W/cm2
Pressures
unknown
130
PFP-Nanobubble
TPPS derivatives
Acted as cavitation nuclei, enhanced sensitizer tumor
penetration
3 MHz
1.8 W/cm2
Pressures
unknown
158
PFC-Nanodroplet
IR780
Enhanced sensitizer tumor penetration via diffusion and
ADV vascular disruption
1.2 MHz
650 kHz
4.5−6.0 W
2.4 W/cm2
Pressures
unknown
159
160
TiO2
TiO2
Alternate Sonosensitization
Metal-based 1O2 generation
1 MHz
0.1−1.2
W/cm2
30 W
1.0 W/cm2
Pressures
unknown
131,133−135,137,138
1.5 MHz
Not listed
132,139
140
AuNPs
Au
Metal-based ROS generation (plasmonic effect)
1.7 MHz
0.008−0.080
mJ/cm2
Pressures
unknown
148
NiFe2O4/C
Graphene
Promotion of ROS production, potential hyperthermia
application
1 MHz
1.0 W/cm2
Pressures
unknown
149
Metal−Organic
frameworks
PMCS
Metal-based ROS generation (gap in between occupied/
unoccupied molecular orbital)
1 MHz
2.5 W/cm2
Pressures
unknown
143
Silica NPs
None
Cavitation nuclei, enhancement of hyperthermia
0.88/2.64
MHz
Not listed
1.0−2.0
W/cm2
2W
Pressures
unknown
151
Rose Bengal
150
PTFE NPs
N/A
Lower cavitation threshold, higher hydroxyl yields
2 MHz
2.0−5.0 MPa
161
HMON + O2
HMME
IR780
Amplifying ROS Generation
Oxygen delivery to hypoxic tumors to improve ROS
generation
3 MHz
1 MHz
5 W/cm2
1 W/cm2
156
144
HMON + TPZ
Chlorin e6
Hypoxia-triggered ROS generation via TPZ
1 MHz
1 W/cm2
145
HMON + Ferrate
(VI)
PpIX
GSH depletion for improved local ROS yield
1 MHz
1.4 W/cm2
Pressures
unknown
56
PtCu3
PtCu3
Act as horseradish-peroxidase-like enzymes, catalyzing
H2O2 into ·HO and as GSH-Px-like enzyme to deplete
GSH and increase ROS for chemodynamic enhanced
SDT
35 kHz
3.0 W/cm2
Pressures
unknown
152
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Table 2. continued
Nanoparticle
Sensitizer
TiO2 + TPZ
TiO2
Fe (III) MON +
SOD2 siRNA
TPPS
Liposome + Dox
Chlorin e6
US
Frequency
Function/Effect
Amplifying ROS Generation
Hypoxia-triggered ROS generation via TPZ
Intensity/
Pressure
Ref
1 W/cm2
Pressures
unknown
157
High ROS efficiency, FL/MR imaging, vehicle to deliver
1 MHz
siRNA, reduction of GSH levels, generation of Fenton for
chemodynamic enhanced SDT
0.56 W/cm2
Pressures
unknown
162
Combination Therapy
SDT-triggered Dox release for SDT + chemo
2 W/cm2
163
1 MHz
1 MHz
Liposome +
HMME
immunoadjuvants
Co-checkpoint-blockade immunotherapy for antimetastatic 1 MHz
response
1.5 W/cm2
146
Liposome
Zinc Phthalocyanine
PDT + SDT
1.1 MHz
1 W/cm2
(ISATA)
Pressures
unknown
164
TiO2+ Dox
TiO2
ROS-triggered Dox release for SDT + chemo
1.5 MHz
3−15 W/cm2
141
TiO2
Black TiO2−x
Enhanced electron−hole separation for enhanced
sonocatalytic efficacy to mediate SDT + PTT
1 MHz
1.5 W/cm2
165
Au-TiO2
TiO2
Enhanced ROS yield and NIR II red shift for improved
SDT + PTT
3 MHz
0.5 W/cm2
Pressures
unknown
166
Polymeric + PTX
IR780
SDT-triggered release for SDT + chemo
1 MHz
0.1−0.4
W/cm2
167
Polymeric + Dox
Chlorin e6
SDT + chemo with slight immunological response
observed
1 MHz
1.0 W/cm2
Pressures
unknown
168
MSNs + curcumin
TiO2
SDT-triggered curcumin release for SDT + chemo
1 MHz
2 W/cm2
Pressures
unknown
169
Dendrimer + Dox
Indocyanine Green
SDT-triggered Dox release for SDT + chemo
1.20 MHz
1−3W
Pressures
unknown
170
AuNPs
Au
Enhanced cell killing observed post-2 Gy X-ray RT + US
1 MHz
0.5−1.5
W/cm2
Pressures
unknown
171
Janus Pt-CuS
TAPP
PTT controls TAPP release and elevates O2 level to
modulate SDT therapeutic efficacy
1 MHz
1.0 W/cm2
Pressures
unknown
172
The mechanism behind such triggered drug release was
touched upon by Wang et al.130 using liposomes loaded with
doxorubicin and pyropheophorbide-lipid, termed Dox-pp-lipo.
When exposed to 0.3 W/cm2 intensity ultrasound, the authors
observed increased doxorubicin release in solution compared
to liposomes devoid of pyropheophorbide. The authors
suggested that ROS generated from pyropheophorbide
sensitization by ultrasound could enable lipid oxidation of
the nanoparticle shell and subsequent release of the loaded
doxorubicin. This was supported by the observation of ·OH
generation in solution following sonication, and reduction in
doxorubicin release following the addition of the 1O2 scavenger
NaN3 to the liposomal solutions. Improved in solution and
intracellular doxorubicin release translated to an increase in
median survival of U87-bearing mice by 23 days when
administered Dox-pp-lipo + ultrasound versus doxorubicin
alone. Based on the sonodynamically dependent on-demand
drug delivery elucidated within the above studies, it is of great
interest within the scope of SDT to further optimize this novel
approach, not only as a means of improving sonosensitizer
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have contributed to the cavitation effect observed. Further
studies confirming the role of nanoconstructs to act as and
stabilize cavitation nuclei have been performed using
mesophorous silica nanoparticles,153 single-cavity polymeric
nanoparticles (nanocup),154 and polytetrafluoroethylene nanoparticles.155 Should nanoparticles indeed serve as nuclei for
acoustic cavitation, they should lower the pressure threshold
required to enable SDT effects, similarly to microbubble
contrast agents. However, nanoparticles hold the added
advantage of being able to extravasate beyond the vasculature.
Thus, nanoparticles hold the capacity of more broadly
augmenting SDT both intra- and extravascularly, regardless
of whether the mechanisms underlying their therapeutic
response is attributable to mechanical shearing, pyrolysis, or
sonoluminescence. This advantage can be further extended
through the use of nanodroplets and nanobubbles as will be
discussed below when considering the future outlook of
nanomedicines in SDT.
3.2.3. Amplifying ROS Generation. In addition to serving as
potential cavitation nuclei, nanoformulations of sonosensitizers
may also amplify the generation of ROS by overcoming or
exploiting tumor hypoxia. This advantage is centered around
the ability of nanoparticles to house multiple drugs within a
single agent. Presuming that a ROS-dependent SDT
mechanism of action predominates observed therapeutic
effects, the codelivery of oxygen with sonosensitizers should
increase ROS generation, especially in hypoxic tumor environments. This indeed has been observed for nanoformulations
combining sonosensitizers and oxygen-carrying perfluorocarbons.144,156 Coloading of oxygen and IR780 within HMONs
proved to increase intracellular ROS production and incited
cell death in otherwise SDT-resistant PANC-1 cells.144
Impressively, this combination SDT/oxygen therapy decreased
tumor hypoxia compared to control tumors and increased the
survival of subcutaneous PANC-1 tumor-bearing mice over a
60-day period compared to SDT conducted without oxygen
coloading (Figure 5c,i-ii). It should be noted that this was one
of the few studies within the field of SDT that demonstrated
such longitudinal therapeutic effects.
Alternatively, it is believed that SDT itself can yield a
hypoxic tumor environment through the consumption of
molecular oxygen during sonochemical reactions between
cavitating bubbles and proximal sonosensitizers. Wang et al.
took advantage of this phenomenon to amplify ROS
generation through the activation of tirapazamine (TPZ), a
hypoxia prodrug that is only activated into its free radical form
under hypoxia.145 The authors coloaded TPZ, Holmium
(imaging agent) and chlorin-e6 (Ce6; sonosensitizer) into
hollow mesoporous silica nanospheres modified with APTES
and conjugated to mAbPSCA for tumor cell targeting. SDT ROS
generation was confirmed only in the presence of Ce6 via
quenchers, reaffirming the necessity of the sonosensitizer for
ROS generation. Both pH- and GSH-responsive controlled
drug release was observed in solution with less than 10%
leakage under normal physiological conditions. This translated
into cytotoxic effects in vitro, reducing PC-3 cell viability to
<20% following nanoparticle and ultrasound treatment. As
postulated by the authors, nanoparticle-mediated SDT created
a hypoxic tumor environment (Figure 5c,iv) that the authors
exploited in vivo to deliver SDT and bioreductive therapy
(BRT). The authors observed 85% tumor growth inhibition by
day 14 after SDT (particles with Ce6 and no TPZ) and 91%
tumor growth inhibition was observed with SDT and BRT
delivery, but also more broadly to improve drug release from
existing chemotherapy nanoparticle formulations. This advancement in the field of SDT would be analogous to PDTtriggered drug delivery, whereby SDT-triggered release holds
the potential of targeting deeper tumor tissues.
3.2.2. New Avenues for Sonosensitization. While current
sonosensitizer design philosophies are centered around the
application of PDT molecular sensitizers, the use of nanomedicine within the field of SDT has opened the door to
delivering sensitization through inorganic nanoparticles.
Inspired by multidisciplinary work in catalytic chemistry,
titanium oxide nanoparticles (TiO2),131 Au deposition on
TiO2 nanoparticles,132 and graphene oxide integration onto
TiO2 nanoparticles133 were applied to facilitate electron−hole
separation to amplify ROS generation following SDT. A U251
glioma cell line exposed to TiO2 and 1.0 MHz 1.0 W/cm2
ultrasound for 50 s showed equal cell toxicity when compared
to 18 J/cm2 UV light irradiation at 5.0 mW/cm2. While
photodynamic toxicity was almost completely inhibited by the
addition of glutathione, a free radical scavenger, suppression of
sonodynamic toxicity was not significantly observed following
glutathione addition.134 This suggested that TiO2 may facilitate
sonosensitization in a manner untraditionally associated with
photoactive sensitizers. Similar toxicity results have also been
shown with C32 human melanoma cells135 and HSC-2 human
carcinoma cells.136 Owing to the low ROS quantum yield of
traditional TiO2 nanoparticles from fast electron−hole
recombination, conjugation and functionalization have also
been explored using pre-S1/S2 antibody recognizing hepatocytes,137 avidin immobilization,138 hydrophilization with
carboxymethyl dextran (CMD),139 and autophagy regulation
loading140 to enhance cellular uptake and increase cellular
concentrations. More recent studies have investigated
combinational sonodynamic chemotherapy using doxorubicin-coordinated TiO2 nanoparticles capable of tumor targeting,
controlled drug release, and intracellular ROS generation.141
Although not directed toward SDT specifically, an in-depth
overview of the conceptual enhancement of cytotoxicity by
TiO2 nanoparticles with exposure to ultrasound has been
explored by Shimizu et al.147 Given these initially promising
results, further exploration of TiO2 nanoparticle SDTmechanistics may yield more efficacious avenues for sonosensitization
and better inform the current understanding of SDT
mechanisms of action. Other inorganic nanoparticles explored
within literature include gold,148 nickel ferrite/carbon,149 iron
oxide,150 silicon,151 platinum,152 and graphene,133 the mechanisms of action of which also need to be better eludicated.
One potential means by which inorganic nanoparticles may
facilitate or augment sonosensitization is by acting as cavitation
nuclei. In 2005, Tuziuti et al. demonstrated that adding
alumina particles to an aqueous solution increased the
presence of harmonics associated with bubble cavitation, and
also increased the temperature of the solution, presumably by
increasing the number of sonochemical hotspots generated142
(Figure 5b,ii). This was followed more recently by Pan et al.143
who developed a metal−organic framework-derived carbon
nanostructure containing porphyrin-like metal centers
(PCMS). Movies captured of the PCMS cavitation effects
demonstrated identical growth and collapse of cavitation
bubbles, but with an increased number and size of cavitation
clusters in PMCS-containing solution when compared to
ultrasound irradiation of water alone (Figure 5b,iii). The
observation of microjets in US-irradiated PMCS solutions may
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combinations have been explored to impede tumor metastasis
and exert synergistic therapy. Treatment of 4T1 xenotransplanted mice with DVDMS (sonosensitizer), 2-deoxyglucose
(glycolysis suppressor), and 1.90 MHz 2 W/cm2 ultrasound
resulted in a slight decrease in pulmonary nodule count when
compared to SDT alone. The effect against metastatic tumor
tissue was attributed to an observed downregulation of both
HK2 and Glut1 expression, leading to reduced levels of
glycolysis in tumor tissue. This prevented cellular proliferation
and metastases through alterations in cellular homeostasis.178
Further studies using a nanocomposite composed of
doxorubicin (chemotherapeutic), indocyanine green (sonosensitizer), and hyaluronic acid (CD44 targeting) demonstrated similar decreases in pulmonary metastasis.170 ROS
production was also confirmed within the SDT group,
potentially implicating its role in the observed antimetastatic
effects. Altogether, while more preclinical models are necessary
to establish the use of SDT as an anti-metastatic treatment,
there does appear to exist preliminary preclinical evidence to
support the application of SDT as an adjuvant therapy that can
potentially prevent the onset of metastatic dissemination.
The reduction in tumor metastasis observed following SDT
may be the result of a cancer cell antigen-mediated immune
response. As such, the concept of an SDT-induced immune
response has been recently studied for SDT/immunotherapy.146,179 In a recent 2019 study, Yue et al. investigated
nanoparticles comprising HMME as the sonosensitizer and
imiquimod as a TLR7 agonist (Figure 5d). Intracellular ROS
generation was confirmed following the application of SDT to
4T1 cells. The subsequent immune response elicited in vitro
and in vivo was mildly enhanced acutely compared to SDT
without imiquimod, as measured through cytokine secretion,
DC maturation, and calreticulin (CRT) exposure. Crucially, no
notable cytokine storm effects (a major toxic limitation of
immunotherapy) were observed. The authors combined the
imiquimod/SDT combination therapy with anti-PDL1 therapy
in vivo in both a local 4T1 and polyclonal whole-body
metastatic model (fLuc-4T1). In both, the SDT/PDL1 therapy
was able to decrease primary and distant tumor growth in
addition to tumor metastasis beyond PDL1 therapy alone.
However, it was unclear whether imiquimod coencapsulation
alongside HMME was necessary to mediate these therapeutic
effects. Nevertheless, the immunomodulatory and therapeutic
effects observed, including the induction of cytokines (TFN-α,
IFN-γ) from TEM CD8+ T cell phenotype expression,
suggests that combination SDT/immunotherapy is a promising
avenue to explore that can capitalize on the coloading of
immunomodulatory agents and sonosensitizers. This offers
great potential as a multifunctional nanoagent for not just
efficacious cytotoxic effects, but also secondary preventative
effects.
In addition to chemo/SDT and immune/SDT, SDT
combination with gene therapy,162 photodynamic therapy,180,181 photothermal therapy,165,166,172 chemodynamic
therapy,152 and radiotherapy171 have also been explored,
yielding a plethora of multifunctional theranostic nanosonosensitizer agents. For example, Shanei et al. used gold
nanoparticles to deliver single-agent SDT (1 MHz, 0.5/1/1.5
W/cm 2) and radiotherapy (X-ray, 0.5/1/2 Gy). 171 A
significant decrease in cell viability was observed when
combination therapy was delivered compared to ultrasound
and radiotherapy alone. While the effect of SDT alone was not
shown, a reduction of cell survival of 94.9% in combination is
(particles containing Ce6 and TPZ). A similar technique of
activating hypoxic prodrugs with SDT was employed by Feng
et al., who loaded TPZ into mesoporous titanium nanoparticles
modified by S-nitrosothiol.157 Akin to the above study,
significant improvements in tumor inhibition were observed
with the addition of the hypoxic prodrug.
Alternative strategies in amplifying ROS generation revolve
around targeting glutathione (GSH) levels within the tumor
microenvironment. The free thiol groups in GSH function to
protect cells against free-radical damage,173 thus compromising
the therapeutic efficacy of ROS-based treatments such as SDT.
Furthermore, GSH also holds the potential to be exploited for
controlled drug release owing to its elevated concentration in
tumor cells.174 Accordingly, the depletion and exploitation of
GSH levels have been recently studied to enhance the efficacy
of SDT-based treatment.152 For example, manganese-based
nanocomposites were explored to both deplete GSH and
generate O2 through H2O2 consumption to overcome the
hypoxic tumor microenvironment. Fu et al. explored
PEGylated K2FeO4 loaded onto hollow mesoporous organosilica nanoparticles (HMON) to which PpIX (sonosensitizer)
was anchored with lauric acid (US-triggered phase change
prodrug platform).56 The authors hoped the inclusion of iron
would potentially enhance ·OH generation via the Fenton
reaction, should that be an underlying mechanism behind
SDT.162 Indeed, the iron oxide inclusion increased both 1O2
and OH· generation, confirmed through fluorescent ROS
probes. A synergistic decrease of cell viability following SDT
treatment (1 MHz, 2 W/cm2) was observed in both hypoxic
and normoxic conditions in both a concentration-dependent
and ultrasound intensity-dependent manner. This translated
into the observation of tumor growth arrest over 18 days only
when SDT was mediated with nanoparticles comprising the
iron oxide and sonosensitizer. Similar manganese-based
nanocomposites explored include PEGylated oxygen-deficient
manganese−tungsten bimetallic nanoparticles for multimodal
image-guided enhanced efficacy SDT, 175 and HMME
(sonosensitizer)-Acriflavine (inhibitor of HIF-1α) encapsulated liposomes coated with MnO2 nanosheets and decorated
with AS1411 aptamer for tumor targeting.176
Thus far, strategies to overcome or exploit tumor hypoxia to
enhance SDT effects are predominantly based on the
coloading of oxygen or hypoxic prodrugs alongside sonosensitizers within nanoformulations. In addition, autophagy
inhibitors have also been employed to induce vessel-normalization,140 and remodeling of tumor-associated macrophage
phenotype (M2 → M1).177 These approaches toward
amplifying ROS generation have only recently been
implemented and thus may warrant further exploration,
including greater characterization of downstream effects and
any potential resistance arising from activation of alternative
antioxidant pathways, such as Trx and NRF2.
3.2.4. Combination Therapies Derived from Multifunctionality. The codelivery of sonosensitizers and hypoxia agents
can be extended to the multifunctional formulation of
nanomedicines containing sensitizers and chemotherapeutics
for synergistic or combination therapy. As overviewed above,
coloading of doxorubicin and sonosensitizers within a single
nanoparticle can facilitate on-demand, externally stimulated
drug delivery. This has since been applied using different
sonosensitizer−doxorubicin combinations163,147 and extended
to the SDT-stimulated delivery of paclitaxel167 and curcumin.169 Beyond its use to trigger drug delivery, chemo/SDT
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pressure reporting, while also combining in solution detection
of soluminescence, broadband emissions associated with
inertial cavitation and ROS generation to characterize their
SDT platform.120,183 The correlation of acoustic emissions in
vivo with SDT bioeffects, though more challenging, can be
guided by existing methodologies implemented in the field of
microbubble-enabled blood-brain barrier disruption summarized recently by Jones and Hynynen.184
4.1.3. Translation of in Vitro Mechanisms to an in Vivo
Setting. When possible, the above techniques should be
applied both in vitro and in vivo. Although in vitro mechanistic
studies evaluating ROS generation have informed our
understanding of SDT, they are not able to account for the
influence of the complex tumor microenvironment, interstitial
and solid tumor pressures, three-dimensional volumetric
boundaries for bubble cavitation, ROS quenchers in tissue,
and light absorbers on cavitational bioeffects. Thus, in vivo
probing of sonosensitizer biodistribution, inertial cavitation,
ROS generation, and sonoluminescence would be valuable in
establishing mechanisms of action. In vivo biodistribution
studies could particularly facilitate delineation between
bioeffects from enhanced drug delivery versus those that are
truly sonodynamic in nature. Specifically, biodistribution of
free sensitizers should be compared to that associated with
nanoformulations to truly differentiate any drug delivery
advantages of nanomedicines. These biodistribution studies
should be performed quanitatively via analytical techniques
such as high-performance liquid chromatography, spectrofluorometry, or inductively coupled mass spectrometry
quantification of sonosensitizers extracted from tissue, or
through radioisotope-enabled quatitative methods such as
positron emission tomography or γ-counting of tissues. In
order to more comprehensively assess whether the bioeffects
observed are a result of cavitation-mediated vasculature
disruption rather than the sonodynamic activation of
sonosensitizers, techniques that assess tumor vascularity can
be employed, such as gadolinium-enhanced T1w-MRI. These
techniques should be combined with sonosensitizer biodistribution analysis across appropriate control groups as will be
discussed below. We do note that the translation of in vitro
characterization of sonoluminescence and ROS generation to
an in vivo setting is challenging. However, we hope that
transdisciplinary collaborations among chemists, physicists,
histologists, and biologists may generate new in vivo probes
and imaging paradigms to address this hurdle. For example,
advances in pairing intravital imaging with ultrasound delivery,
cavitation detection, and the development of novel fluorescent
and chemiluminescent probes may be of value in garnering
SDT mechanistic insights in vivo. Furthermore, acute
histological analysis of tumor tissue could further help
distinguish bioeffects and resulting mechanisms of action,
including differentiation of expected SDT cell apoptosis from
thermally induced tissue necrosis and histotripsy tissue
fractionation.
4.1.4. Use of Appropriate Controls. The current broad
definition of SDT involves the use of low frequency ultrasound
to activate therapeutic effects of sonosensitizers. Though
synergy between ultrasound and sonosensitizer is often
implied, this synergy can only be demonstrated by incorporating adequate controls into therapeutic studies. Specifically, this
broad definition and implication of synergy necessitates at
minimum the comparison of therapeutic effects associated with
(i) ultrasound alone, (ii) sonosensitizer alone, and (iii)
nonetheless highly promising. Furthermore, a SDT/radiation
therapy strategy can capitalize upon existing platforms that
have already been explored clinically to deliver HIFU therapy
in conjunction with low-dose radiotherapy.182 Indeed, all of
the above explored SDT combination therapy paradigms can
benefit from advances in the implementation of individual
therapies, leading to potentially synergistic effects that can
augment the therapeutic capacity of SDT.
4. FUTURE OUTLOOK
4.1. Overcoming Current Limitations. Despite the
advantages that nanomedicine has brought to SDT, ambiguity
surrounding the mechanisms of action at play creates
limitations in maximizing therapeutic efficacy. This is further
challenged by a lack of standardization in ultrasound dosimetry
and a unifying definition of SDT. To overcome these
challenges, we propose reporting acoustic intensities in the
form of pressures, assessing ultrasound acoustic emissions
during SDT, detecting sonoluminescence, quantifying sensitizer biodistribution, assessing tumor vasculature permeability,
and employing appropriate treatment controls as discussed
below.
4.1.1. Standard Reporting of Ultrasound Parameters. As
discussed, cavitational bioeffects depend largely on acoustic
pressure. It is therefore peculiar that acoustic pressures are
rarely reported in SDT studies. Instead, authors typically report
acoustic intensities in the form of power density (W/cm2).
This limits the comparisons that can be made between studies
and does not provide enough context to delineate whether the
pressure pulses fall within realms of stable or inertial cavitation.
While pressure−intensity conversions may be estimated using
the formula I = p2/ρc, where I = instantaneous acoustic
intensity, p = root-mean-square pressure amplitude, ρ =
density of the propagating medium, and c = velocity of sound
in the propagating medium, significant assumptions must be
made to derive pressures in this manner since each ultrasound
transducer’s output power density corresponds to differing
pressures. As such, authors should ensure that they calibrate
their ultrasound systems and transducers to more accurately
report acoustic intensity in the form of pressure, and outline
their ultrasound setups and beam characteristics in more detail.
We hope that the combined reporting of frequency, beam
geometry, ultrasound setup, and pressure becomes standard so
that lessons learned from individual studies can be more
broadly applied.
4.1.2. Better Characterization of Cavitation and Resultant
Effects. As ROS generation is the predominating therapeutic
mechanism of action used to explain SDT effects, there exists
an underlying assumption that inertial cavitation is a
prerequisite of SDT. However, few studies directly provide
evidence of inertial cavitation, or any resulting hotspot
temperature changes and sonoluminescence. In order to better
elucidate SDT mechanisms, acoustic cavitation, sonoluminescence, and temperature detection within SDT studies would be
of great value. This is particularly vital in evaluating the
therapeutic utility of nanoparticles in SDT: cavitation
detection over a range of pressures will better differentiate
whether nanoparticles augment SDT effects by lowering
cavitation thresholds as exogenous sources of cavitation nuclei,
or by other mechanistic means such as improving sonosensitizer bioavailability or activity. To this end, lessons can be
learned from recent studies by Beguin et al., who thoroughly
described their acoustic instrumentation, including acoustic
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include nanobubbles, echogenic liposomes, gas vesicles, and
cavitation seeds.187 Echogenic liposomes comprising gas
pockets within the lipid bilayer or core monolayers may
particularly be of interest, as they may facilitate higher
sensitizer loading versus nanobubbles or droplets.187,188
The above submicron SDT strategies are advantageous for
tumors displaying the enhanced permeability and retention
(EPR) effect, whereby the rapid growth of tumors leads to
leaky vasculature, allowing nanoparticle extravasation followed
by retention due to impaired lymphatic drainage. Tumors that
are not susceptible to the EPR effect, such as blood-brain
barrier intact glioblastomas, would require alternative strategies
not reliant on the EPR effect for nanomedicine delivery. One
such example is the use of an acoustic microbubble-tonanobubble conversion, introduced by Hunyh et al.189 Here,
bacteriochlorophyll−lipid shelled microbubbles underwent an
in situ conversion into nanoparticles following the application
of low frequency therapeutic ultrasound. This led to early
sequestration of the resulting daughter nanoparticles within KB
subcutaneous tumors in mice, potentially from vasculature
disruption at the tumor site. Electron microscope imaging
indicated that this conversion likely produced nanobubbles as
opposed to gas-devoid nanoparticles. Presumably, this microto-nano conversion could be exploited to deliver nanobubbles
across intact tumor vasculature, where the bubbles could then
be stimulated to cavitate and activate the loaded sonosensitizer
(in this case, bacteriochlorophyll, a sister compound to
porphyrin) for extravascular SDT. However, prior to realizing
this potential, confirmation of vasculature disruption and the
delivery of nanobubbles through this micro-to-nano conversion
is required.
4.3. Bearings on Clinical Translation. The ultimate goal
of improving and optimizing any therapeutic paradigm
preclinically, including SDT, is translation to the clinic.
Despite the myriad in vivo SDT studies, clinical trial validation
remains to be sought. While clinical case studies report initial
promise,190,191 only combination therapy with PDT180 or
immunotherapy has been explored for cancer treatment,
making it difficult to elucidate the true independent clinical
effects of SDT. The largest of such studies featured 115
advanced metastatic patients unresponsive to traditional
therapy with varying primary tumor sites.180 These patients
received combinatorial sono-photodynamic therapy (1 MHz, 1
W/cm2) with Sonnelux-1 under generalized light and showed
an overall increase of predicted median survival. Although
preliminarily promising, it remains difficult to evaluate the
study in depth given the lack of statistical analysis conducted
with regard to overall survival benefit and a selective
representation of patient data in favor of those that showed
improvement to predicted median survival. Furthermore, no
information regarding the ultrasound apparatus was provided.
Given the usage of ultrasonic irradiation in a bathtub as well as
other suboptimal unfocused ultrasound platforms for SDT
treatment in previous case studies,190 this lack of information is
troubling.
The limited clinical application of SDT is unsurprising. The
SDT field is still young and, as discussed, requires further
exploration before realizing its promising clinical potential as a
minimally invasive, safe, and targeted adjunctive cancer
therapy. This limited clinical exploration underscores the
need to develop a greater understanding of the mechanisms
underlying SDT, but also more detailed exploration beyond
proof-of-concept studies of existing therapeutic regimens that
combination of ultrasound and sonosensitizer. Additionally,
synergy should be calculated and not assumed in order to
distinguish from additive effects. This is particularly of
importance when assessing SDT in combination with other
therapies, such as PDT, chemotherapy, PTT, and others. The
implementation of ultrasound-only controls when combined
with tumor vessel permeability assessment and thermometry
will additionally allow the contribution of thermal ablation,
hyperthermia, and vasculature disruption toward observed
bioeffects to be better assessed. To this end, MRI thermometry
during SDT can provide valuable information as evidenced by
Wu et al.35 Furthermore, the implementation of micron and
nanosized agents in SDT additionally requires the comparison
of free and encapsulated sonosensitizer with and without
ultrasound administration to truly realize the therapeutic
beneficence of using supramolecular variants of sonosensitizers. Lastly, given the debate over whether sonosensitizers
function as photodynamic agents, amplifiers of pyrolysis, or
mediators of mechanical bioeffects, the photodynamic activity
of sonosensitizers should be characterized if unknown.
Together, these controls can better differentiate bioeffects
resulting from microbubble cavitation versus sonosensitizer
activation.
4.1.5. Generating Sensitizer-Specific Structure/Activity
Relationships. Collectively, the application of the above
recommendations across different classes of sensitizers may
better clarify SDT structure/activity relationships. We hope
these studies reevaluate seminal disseminations and previously
studied sensitizers using advances in ROS probe chemistry,
acoustic cavitation detection, and ultrasound transducer design
to better clarify past contradicting evidence surrounding SDT
mechanisms of action. This demanding task would allow better
optimization of sonosensitizers, elucidation of nanomedicine
benefits, and more informed selection of synergistic therapy
paradigms.
4.2. Expanding the Unique Role of Nanomedicine.
The established utility of nanomedicine in SDT, as overviewed
above, can be further expanded through the use of submicron
ultrasound contrast agents. These nanosized echogenic
particles may function similarly to microbubbles as exogenously delivered acoustically responsive nuclei that may lower
the threshold for acoustic cavitation required for SDT. Unlike
limitations faced by their micron-sized counterparts, nanosized
contrast agents have the ability to extravasate out of leaky
tumor vasculature into the tumor interstitium, with optimal
nanoparticle sizes for passive tumor uptake thought to be in
the 100−150 nm size range.185,186 This could allow for more
efficient SDT to be conducted within both the intravascular
and extravascular tumor spaces, expanding the therapeutic
horizons for SDT and allowing for spatially varied SDT effects
to be better studied, a feat unachievable with micron-sized
contrast agents. The potential of sensitizer-loaded, nanosized
ultrasound contrast agents to act as SDT sensitizers remains
untapped, with merely a single in vivo160 study published in
2019 to the best of our knowledge that made use of echogenic
nanoparticles. In this study, nanodroplets were used to deliver
IR780 sensitizer beyond the tumor vasculature and incite SDT
in 4T1 tumor-bearing mice. Thus, there is much to still be
learned about the use of nanosized ultrasound contrast agents
as extravascular agents, including whether they can lower the
cavitation threshold of SDT, induce more potent effects versus
nonechogenic nanoparticles, deliver extravascular SDT, and
extend to echogenic nanoparticles beyond nanodroplets to
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field in establishing universal mechanisms of SDT, innovation
continues in the field. Promoted by advances in nanotechnology, numerous novel nanoplatforms have been
developed to better enhance the therapeutic efficacy of SDT.
Through enhanced spatiotemporal accumulation of single or
multiple drug agents in high packing densities combined with
their ability to function as cavitation nuclei, nanoscaled
sonosensitizers may overcome the barriers observed thus far
in achieving therapeutically effective SDT. Thus, when
combined with advances in the clinical implementation of
low frequency focused ultrasound, and better mechanistic
clarity, nanomedicines may bring the field of SDT closer to
realizing its exciting therapeutic potential.
have already shown initial preclinical success. To better
illuminate the necessity of establishing more detailed
mechanistic understanding, one needs only to look within
this decade toward latrepirdine, an antihistamine proposed for
usage in Alzheimer’s disease. Similar to SDT, preclinical
studies showed very promising results in vitro, although no
investigation was undertaken to comprehensively evaluate the
mechanism by which such effects were exerted.192 This
inevitably proved to be its downfall, as three pivotal phase
III clinical trials all showed negligible cognitive benefits, and
only recently has data contradicted previously held hypotheses
on latrepirdine mechanistics.193 It is true that for a drug to gain
FDA approval, understanding of mechanisms is not strictly
required, and indeed, the mechanisms of action of many highly
prescribed drugs remain unknown. Nevertheless, this knowledge, particularly when considering a novel therapeutic
modality such as SDT, guides drug development and is
essential in providing direction by which the field may grow.
As such, the formation of a foundational understanding of
SDT will be the first step toward its effective clinical
translation. In understanding SDT, the field has a greater
chance of overcoming hurdles in delivering effective SDT
preclinically, without which clinical trial of SDT is inherently
impeded. Once consistent, effective SDT is achieved preclinically; its successive clinical translation will then also depend on
its rational application to appropriate cancer lesions. SDT
translation will also require advances in the clinical
implementation of therapeutic focused ultrasound, including
MRI-guidance and more readily available MRI and patientcompatible focused ultrasound transducers. Finally, the success
of clinical SDT will also be contingent on the effective
translation of sonosensitizers and sonosensitive nanomedicines. Thus, intrinsically, the challenging task of realizing the
therapeutic potential of SDT will require collaboration across
multiple disciplines.
■
AUTHOR INFORMATION
Corresponding Author
Gang Zheng − Princess Margaret Cancer Centre, University
Health Network, Toronto, Ontario, Canada M5G 1L7;
Institute of Biomaterials and Biomedical Engineering and
Department of Medical Biophysics, University of Toronto,
Toronto, Ontario, Canada M5S 3G9; orcid.org/00000002-0705-7398; Email: gang.zheng@uhnres.utoronto.ca
Authors
Victor Choi − Princess Margaret Cancer Centre, University
Health Network, Toronto, Ontario, Canada M5G 1L7; School
of Pharmacy, University College London, London, United
Kingdom WC1N 1AX
Maneesha A. Rajora − Princess Margaret Cancer Centre,
University Health Network, Toronto, Ontario, Canada M5G
1L7; Institute of Biomaterials and Biomedical Engineering,
University of Toronto, Toronto, Ontario, Canada M5S 3G9
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.bioconjchem.0c00029
Author Contributions
5. CONCLUSIONS
It is well-known that first-line and adjunct treatment modalities
for cancer, including chemotherapy, ionizing radiation, and
surgical resection, are invasive, immunosuppressive, or
cumulatively toxic. Therefore, the promise of a new therapeutic
option that can access and treat deep-seated tumor lesions in a
minimally invasive manner using relatively nontoxic agents
warrants the broader exploration of SDT as a cancer therapy.
While a growing amount of research has been conducted
preclinically, a deeper understanding of the mechanisms
underlying SDT is needed for the field to realize its potential.
These mechanisms include mechanical cell membrane shearing
and ROS-mediated cytotoxicity from pyrolysis or sonoluminescence, wherein ultrasound-triggered, sonosensitizer-enabled
ROS generation currently stands as the prevailing proposed
mechanism of action. These are all plausible contributors to
therapeutic efficacy, whereby the predominating mechanism is
likely dependent on the physiochemical properties of
sonosensitizers, ultrasound dosimetry, and sensitizer intratumoral and subcellular localization. While it is unreasonable
to expect a single comprehensive study analyzing each
combination of these variables, we hope that standard
reporting and characterization of ultrasound dosimetry, inertial
versus stable cavitation, in vivo mechanistic studies, and the use
of appropriate therapeutic controls can generate transferrable
knowledge to better inform the design and implementation of
SDT paradigms. Nevertheless, despite challenges faced by the
#
This manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript. V.C. and M.A.R. contributed equally.
Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS
The authors would like to thank Alex Dhaliwal and Carly
Pellow for their technical expertise. The authors acknowledge
their funding sources: the Terry Fox Research Institute, the
Canadian Institutes of Health Research, the Natural Sciences
and Engineering Research Council of Canada, the Princess
Margaret Cancer Foundation, Canada Research Chairs
Program and the McLaughlin Centre.
■
ABBREVIATIONS
CMD, carboxymethyl dextran; DOX, doxorubicin; DVDMS,
sinoporphyrin sodium; EPR, enhanced permeability and
retention; GSH, glutathione; HIF-1α, hypoxia inducible factor
1-alpha; HIFU, high intensity focused ultrasound; HMME,
hematoporphyrin monomethyl ether; HMONs, hollow mesoporous organosilica nanoparticles; IFN-γ, interferon gamma;
MDRP1, multidrug resistance protein 1; MMP, mitochondrial
membrane potential; MRI, magnetic resonance imaging; MSN,
mesoporous silica nanoparticle; NRF2, nuclear factor erythroid
2-related factor 2; NSAIDs, nonsteroidal anti-inflammatory
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drugs; PCMS, porphyrin-like metal centers; PD-L1, programmed death-ligand 1; PDT, photodynamic therapy; PEG,
polyethylene glycol; PFC, perfluorocarbon; PFP, perfluoropropane; PpIX, protoporphyrin IX; PTFE, polytetrafluoroethylene; PTX, paclitaxel; RB, Rose Bengal; ROS, reactive
oxygen species; RT, radiotherapy; SDT, sonodynamic therapy;
TAPP, tetra-(4-aminophenyl) porphyrin; TLR7, toll-like
receptor 7; Trx, thioredoxin reductase; TPZ, tirapazamine
■
Review
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